HIF-1 (hypoxia inducible factor-1) is a protein that plays an important role in various cell responses in hypoxic condition including regulation of a gene involved in energy metabolism, vasomotor control, angiogenesis and apoptosis. In particular, HIF-1α (alpha) is degraded by proteosome under normoxic conditions, while being stabilized in hypoxic conditions. Such mechanism is regulated by the interaction between pVHL (von Hippel-Lindau tumor suppressor) protein and HIF-1α. The inhibition of the interaction between HIF-1α and pVHL results in the acceleration of cell cycle, angiogenesis and cell survival function in hypoxic condition, which provides an idea for the treatment of ischemia such as coronary insufficiency, cerebral insufficiency and vascular insufficiency (U.S. Pat. No. 6,787,326 B1). On the contrary, the promotion of the interaction results in the inhibition of angiogenesis in normoxic conditions, suggesting that tumors might be suppressed by that mechanism (Amato G. et al., Nature Reviews 2: 1-9, 2003).
The binding of VHL protein with human HIF-1α depends on hydroxyproline group residing in 402nd or 564th amino acid of human HIF-1α. And the interaction between VHL protein and HIF-1α is regulated by hydroxylation of a specific proline group of HIF-1α by prolyl hydroxylase (Masson N. and Ratcliffe P J., Journal of Cell Science 116: 3041-3049, 2003). Particularly, prolyl hydroxylase-2 uses oxygen, iron and 2-oxoglutarate as cofactors for its reaction, and at this time ascorbate is necessarily used to prevent rapid inactivation of the enzyme by self-oxidation (Ivan M. et al., PNAS 99(21): 13459-13464, 2002). The disclosure of a mechanism that regulates hydroxylation-induced transcription and proteolytic destruction can be helpful for the studies on pathological mechanisms.
VHL protein is composed of β domain containing about 100 amino acids and α domain containing approximately 35 amino acids. The β domain is bound to Elongin C protein to form a VHL-Elongin C complex and then Elongin B protein is bound to the position of Elongin C to form a VBC protein. To the rest α area of the VBC protein is bound to HIF-1α, during which hydrogen bonds between the 115th His group and the 111th Ser group of VHL protein and the 564th hydroxy-Pro group of HIF-1α are crucial (Min J. H. et al., Science 296: 1886-1889, 2002).
The interaction between VHL protein and HIF-1α has been detected by biochemical or immunological approaches. First, two-hybrid assay has been used to detect the interaction between two different proteins. In this assay, DNA binding domain (DBD) of yeast GAL4 transcription factor is fused into VHL protein and transcriptional activation domain (TAD) is fused into HIF-1α, respectively, and then the transcription is activated by the binding of the two proteins. At this time, the activation of the transcription induced by the binding of the two different proteins is easily detected by using a reporter gene such as GFP (green fluorescent protein), luciferase and β-galactosidase. That is, upon the combination of VHL protein and HIF-1α, a reporter gene is activated, by which the interaction between heterologous proteins can be easily detected (U.S. Pat. No. 6,787,326 B1).
There is another method to detect the interaction between two proteins, in which either VHL protein or HIF-1α protein is labeled with detectable material and the remaining one is fixed in solid carrier. And the detectable marker is not limited as long as it can be inserted in a recombinant protein, which is exemplified by 35S-methionine, HA tag, GST tag, histidine tag, etc (U.S. Pat. No. 6,787,326 B1). In general, HIF-1α is labeled with GST tag and VHL protein is marked by 35S, and the interaction between the two proteins is observed by SDS-PAGE and autoradiography (Yu F. et al., PNAS 98(17): 9630-9635, 2001). However, this method has problems of requiring a large amount of samples, intricacy of the protocol, lengthy analysis time and the use of radioactive reagents.
Coimmunoprecipitation of VHL protein and a specific part of HIF-1α is a way to screen HIF-1α interacting with VHL protein (Yu F. et al., Cancer Research 61: 4136-4142, 2001). In addition, scintillation proximity assay can be also used to detect the interaction, in which a target compound is radio-labeled and scintillation occurring during the process of binding of two proteins is detected (U.S. Pat. No. 6,787,326 B1).
However, all these biochemical, immunological or radiographic assay present problems including intricacy of protocol and high costs. Therefore, an alternative assay which is easier and simpler is required to observe characteristics of the binding of VHL protein with HIF-1α. Accordingly, a method has been developed that analyzes the binding of VBC protein with HIF and characteristics of hydroxylation involved therein using a 96-well plate in a short period of time without using a radioactive agent (F. Oehme, et al., Analytical Biochemistry 330: 74-80, 2004). Precisely, the method is to quantify OD450 of the complex of biotin labeled HIF-1α (biotinyl-HIF-1α) and VBC protein in a plate coated with avidin. This method enables the observation of hydroxylation of HIF at the concentration range of tens of nano mols and simple quantification, but still presents a problem of requirement of using an expensive avidin coated plate.
Thus, the present inventors have made every effort to develop an alternative assay for quantifying the interaction between HIF-1 peptide and VBC protein, and as a result, the inventors have developed a method for quantitative analysis of the interaction between probe prepared by attaching a fluorescein to hydroxyproline containing HIF-1 peptide and VBC protein using fluorescence polarization. The method of the invention does not require the separation of the binders, making the procedure simple and lowering the costs for analysis owing to the automation using a well-plate. Therefore, the developed assay can be very useful for the detection of an inhibitor of the interaction or for the analysis of the activity of prolyl hydroxylase mediating hydroxylation of HIF-1.